1. Field of the Invention
The invention is directed to purified and isolated novel DAKAR (death-associated kinase containing ankyrin repeat) polypeptides and fragments thereof, the nucleic acids encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, fragmented peptides derived from these polypeptides, and uses thereof.
2. Description of Related Art
Cell death occurs by one of two mechanisms, necrosis or apoptosis. Necrosis is uncontrolled cell death which usually results from environmental stresses such as severe trauma to cells. Cell death from apoptosis, however, results from specific internal signals that activate a cell""s death program. Therefore, apoptosis is termed programmed cell death.
The morphological changes that occur during apoptosis are characterized by DNA degradation by endonucleases, cytoplasmic and nuclear condensation, and the formation of membrane xe2x80x9cblebsxe2x80x9d or apoptotic bodies (T. G. Cotter et al., Anticancer Res, September-October: 10(5A):1153-9, 1990). Neighboring cells then move in to engulf the remaining cellular debris. Programmed cell death has been observed during early development and in immune responses where cells, such as lymphocytes, are eliminated via apoptosis when they are no longer needed (C. A. Janeway, Jr. and P. Travers, Immuno Biology G:3 (Garland Publishing Inc., 2nd ed. 1996)).
The biochemical mechanism driving apoptosis begins with a ligand/receptor induced signal that activates (in part through phosphorylation or dephosphorylation) other proteins, such as kinases, along the signal transduction pathway and ultimately concludes with the activation of the cell death program. Such ligand/receptor pairs that can induce apoptosis are, for example, TNF/TNF-RI, TNF/TNF-R2, CD95 ligand/CD95, TRAIL/TRAIL-R1, and TRAIL/TRAIL-R2.
Many of the receptors above and the intracellular kinases that play a role in transducing the signal from the membrane to the nucleus contain a stretch of 80 amino acids that is necessary to activate cell death. This xe2x80x9cdeath domainxe2x80x9d (DD) functions by interacting with other proteins via their DD or via self association. Over expression of many DD-containing proteins results in cell death, indicating that these proteins may play a role in apoptosis (K. Schulze-Osthoff et al., Eur. J. Biochem, 254:439-459, 1998).
Among the proteins involved in apoptosis are the eukaryotic protein kinases (e.g., cell death related kinases, cell proliferation related kinases, etc.). These kinases make up a large and rapidly expanding family of proteins related on the basis of homologous catalytic domains. Spurred by the development of gene cloning and sequencing methodologies, distinct protein kinase genes have been identified from a wide selection of invertebrates and lower eukaryotes, including Drosophila, Caenorhabditis elegans, Aplysia, Hydra, Dictyostelium, and budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. Homologous genes have also been identified in higher plants. Protein kinases, however, are not limited to the eukaryotes. Enzyme activities have been well documented in prokaryotes, but the prokaryotic protein kinase genes are not obviously homologous to those of the eukaryotes.
Given the important function of kinases in general and DAKAR specifically, there is a need in the art for additional members of the kinase family. There is also a need in the art for the identity and function of proteins having linase activities. Moreover, given the important roles kinases may play in apoptosis, there is an unmet need for therapeutic compounds which interfere with apoptosis.
In another aspect, the identification of the primary structure, or sequence, of an unknown protein is the culmination of an arduous process of experimentation. In order to identify an unknown protein, the investigator can rely upon a comparison of the unknown protein to known peptides using a variety of techniques known to those skilled in the art. For instance, proteins are routinely analyzed using techniques such as electrophoresis, sedimentation, chromatography, sequencing and mass spectrometry.
In particular, comparison of an unknown protein to polypeptides of known molecular weight allows a determination of the apparent molecular weight of the unknown protein (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)). Protein molecular weight standards are commercially available to assist in the estimation of molecular weights of unknown protein (New England Biolabs Inc. Catalog:130-131, 1995; J. L. Hartley, U.S. Pat. No. 5,449,758). However, the molecular weight standards may not correspond closely enough in size to the unknown protein to allow an accurate estimation of apparent molecular weight. The difficulty in estimation of molecular weight is compounded in the case of proteins that are subjected to fragmentation by chemical or enzymatic means, modified by post-translational modification or processing, and/or associated with other proteins in non-covalent complexes.
In addition, the unique nature of the composition of a protein with regard to its specific amino acid constituents results in unique positioning of cleavage sites within the protein. Specific fragmentation of a protein by chemical or enzymatic cleavage results in a unique xe2x80x9cpeptide fingerprintxe2x80x9d (D. W. Cleveland et al., J. Biol. Chem. 252:1102-1106, 1977; M. Brown et al., J. Gen. Virol. 50:309-316, 1980). Consequently, cleavage at specific sites results in reproducible fragmentation of a given protein into peptides of precise molecular weights. Furthermore, these peptides possess unique charge characteristics that determine the isoelectric pH of the peptide. These unique characteristics can be exploited using a variety of electrophoretic and other techniques (T. D. Brock and M. T. Madigan, Biology of Microorganisms 76-77 (Prentice Hall, 6d ed. 1991)).
Fragmentation of proteins is further employed for amino acid composition analysis and protein sequencing (Matsudiara, J. Biol. Chem. 262:10035-10038, 1987; C. Eckerskorn et al., Electrophoresis 1988, 9:830-838, 1988), particularly the production of fragments from proteins with a xe2x80x9cblockedxe2x80x9d N-terminus. In addition, fragmented proteins can be used for immunization, for affinity selection (R. A. Brown, U.S. Pat. No. 5,151,412), for determination of modification sites (eg. phosphorylation), for generation of active biological compounds (T. D. Brock and M. T. Madigan, Biology of Microorganisms 300-301 (Prentice Hall, 6d ed. 1991)), and for differentiation of homologous proteins (M. Brown et al., J. Gen. Virol. 50:309-316, 1980).
In addition, when a peptide fingerprint of an unknown protein is obtained, it can be compared to a database of known proteins to assist in the identification of the unknown protein using mass spectrometry (W. J. Henzel et al., Proc. Natl. Acad. Sci. USA 90:5011-5015, 1993; D. Fenyo et al., Electrophoresis 19:998-1005, 1998). A variety of computer software programs to facilitate these comparisons are accessible via the Internet, such as Protein Prospector (Internet site: prospector.uscf.edu), MultiIdent (Internet site: www.expasy.ch/sprot/multiident.html), PeptideSearch (Internet site: www.mann.embl-heiedelberg.de...deSearch/FR_PeptideSearch Form.html), and ProFound (Internet site: www.chait-sgi.rockefeller.edu/cgi-bin/prot-id-frag.html). These programs allow the user to specify the cleavage agent and the molecular weights of the fragmented peptides within a designated tolerance. The programs compare these molecular weights to protein molecular weight information stored in databases to assist in determining the identity of the unknown protein. Accurate information concerning the number of fragmented peptides and the precise molecular weight of those peptides is required for accurate identification. Therefore, increasing the accuracy in determining the number of fragmented peptides and their molecular weight should result in enhanced likelihood of success in the identification of unknown proteins.
In addition, peptide digests of unknown proteins can be sequenced using tandem mass spectrometry (MS/MS) and the resulting sequence searched against databases (J. K. Eng, et al., J. Am. Soc. Mass Spec. 5:976-989 (1994); M. Mann and M. Wilm, Anal. Chem. 66:4390-4399 (1994); J. A. Taylor and R S. Johnson, Rapid Comm. Mass Spec. 11:1067-1075 (1997)). Searching programs that can be used in this process exist on the Internet such as Lutefisk 97 (Internet site: www.Isbc.com:70/Lutefisk97.html), and the Protein Prospector, Peptide Search and ProFound programs described above. Therefore, adding the sequence of a gene and its predicted protein sequence and peptide fragments to a sequence database can aid in the identification of unknown proteins using tandem mass spectrometry.
Thus, there also exists a need in the art for polypeptides suitable for use in peptide fragmentation studies, for use in molecular weight measurements, and for use in protein sequencing using tandem mass spectrometry.
The invention aids in fulfilling these various needs in the art by providing isolated Death Associated Kinase containing Ankyrin Repeats (xe2x80x9cDAKARxe2x80x9d) nucleic acids and polypeptides encoded by these nucleic acids. Particular embodiments of the invention are directed to an isolated DAKAR nucleic acid molecule comprising the DNA sequence of SEQ ID NO:1 and an isolated DAKAR nucleic acid molecule encoding the amino acid sequence of SEQ ID NO:2, as well as nucleic acid molecules complementary to these sequences. The invention also encompasses recombinant vectors that direct the expression of the nucleic acid molecules of the invention and host cells stably or transiently transformed or transfected with these vectors.
In addition, the invention encompasses methods of using the nucleic acid noted above to identify nucleic acids encoding proteins having kinase activity or proteins involved in apoptosis signal transduction.
The invention also encompasses isolated polypeptides and fragments thereof encoded by these nucleic acid molecules including soluble polypeptide portions of SEQ ID NO:2. The invention further encompasses methods for the production of these polypeptides, including culturing a host cell under conditions promoting expression and recovering the polypeptide from the culture medium. Especially, the expression of these polypeptides in bacteria, yeast, plant, insect, and animal cells is encompassed by the invention.
In general, the polypeptides of the invention can be used to study cellular processes such as immune regulation, cell death, cell migration, cell-to-cell interaction, and inflammatory responses. In addition, these polypeptides can be used to identify proteins associated with DAKAR kinases.
In addition, the invention includes assays utilizing these polypeptides to screen for potential inhibitors of DAKAR activity. Further, methods of using these polypeptides in the design of inhibitors thereof are also an aspect of the invention.
The invention further provides a method for using these polypeptides as molecular weight markers that allow the estimation of the molecular weight of a protein or a fragmented protein, as well as a method for the visualization of the molecular weight markers of the invention thereof using electrophoresis. The invention further encompasses methods for using the polypeptides of the invention as markers for determining the isoelectric point of an unknown protein, as well as controls for establishing the extent of fragmentation of a protein. Further encompassed by this invention are kits to aid in these determinations.
Further encompassed by this invention is the use of the DAKAR nucleic acid sequences, predicted amino acid sequences of the polypeptide or fragments thereof, or a combination of the predicted amino acid sequences of the polypeptide and fragments thereof for use in searching an electronic database to aid in the identification of sample nucleic acids and/or proteins.
Isolated polyclonal or monoclonal antibodies that bind to these polypeptides are also encompassed by the invention, in addition the use of these antibodies to aid in purifying the DAKAR polypeptide and to inhibit apoptosis.